Amplification of chromosome 11q13 DNA sequences is detected in approximately 30% of primary head and neck squamous cell carcinomas (HNSCC). The amplified region includes genes for cyclin D1, hst-1, int-2, and more recently, ems-1. Ems-1 encodes an 80/85kd cytoskeletal associated protein termed cortactin, which has been shown to bind F-actin and is a pp60src substrate. We investigated 16 HNSCC cell lines for ems-1 DNA amplification and gene expression by western blotting and immunofluorescence using mAb 4F11. Amplification of ems-1 DNA was detected in 8/16 (50%) cell lines and was related directly to over-expression of cortactin by western blotting and immunofluorescence. Western blotting detected both forms of cortactin, p80 and p85, at equal intensity. Immunofluorescent staining revealed low levels of cortactin localized to the cytoplasm and surface membrane in normal bronchial epithelial cells and tumor cell cultures with single copy ems-1 DNA. In contrast, tumor cell cultures with ems-1 DNA amplification demonstrated intense, homogeneous cortactin cytoplasmic staining. These results suggest that overexpression of p80/85 may be a useful marker to identify 11q13 amplification, a molecular alteration correlated with the presence of lymph node metastasis in head and neck cancer.